Interakcija proteina Usp bakterije Escherichia coli z evkariontskimi celicami

Zalokar, Danijela (author), Žgur Bertok, Darja (mentor)

Escherichia coli (E. coli) je izredno raznolik organizem. Je komenzal, ki naseljuje prebavni trakt vretenčarjev in je sestavni del njihove mikrobiote. Po drugi strani pa je ta bakterija pomemben patogeni organizem, ki povzroča črevesne in izvenčrevesne okužbe. Med izvenčrevesnimi patogenimi E. coli so zelo pogoste uropatogene E. coli (UPEC). UPEC pretežno uvrščamo v filogenetsko skupino B2 in so odgovorne za kar 80 % okužb urinarnega trakta. Večina ima zapis za Usp – uropatogeni specifični protein. Usp je homologen nukleaznim piocinom in kolicinom in ima in vitro DNazno aktivnost. Predvidevali so, da je bakteriocin, a se je izkazalo, da nima bakteriocidnega učinka na bakterije, ima pa genotoksičen vpliv na evkariontske celice – torej je Usp genotoksin. Navzdol od usp se nahajajo trije geni z zapisom za proteine Imu. Ti imajo vlogo zaščite producenta pred nukleaznim delovanjem Usp. Imu3 reverzibilno veže DNA in promovira genotoksičnost proteina Usp. Cilj magistrske naloge je bila priprava fuzijskega rekombinantnega proteina Usp-GFP, katerega aktivnost bo primerljiva aktivnosti nativne oblike proteina Usp. Optimizirali smo pridobivanje Usp-GFP. Sev E. coli BL21 (DE3) pLyseE transformiran s pTHCG12U smo gojili pri 30 °C, produkcijo proteina Usp-GFP pa smo inducirali z 0,1 mM IPTG. Protein smo izolirali s pomočjo Ni-NTA afinitetne kromatografije, ga očistili s FPLC ter nato preverili njegovo aktivnost. Pripravili smo še izolate Usp in Imu3. Izkazalo se je, da ima Usp-GFP DNazno aktivnost in vpliv na količino biomase HUVEC celic. Njegova in vitro in in vivo aktivnost je primerljiva aktivnosti nativne oblike Usp. Usp-GFP je uporabno orodje za nadaljnje raziskave interakcije proteina Usp z evkariontskimi celicami.

• Language: Slovenian
• Keywords: Escherichia coli, uropatogeni specifični protein, Usp, genotoksin, izolacija proteina, protein Imu3, Usp-GFP
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2018
• PID: 20.500.12556/RUL-104865
• COBISS.SI-ID: 4928847
• Publication date in RUL: 13.10.2018

Secondary language

• Language: English
• Title: Interaction of the Escherichia coli Usp protein with eucaryotic cells

Abstract

Escherichia coli (E. coli) is an extremely versatile organism. It is a commensal bacterium that inhabits the gastrointestinal tract of vertebrates and is a crucial part of their microbiota. On the other hand, this bacterium is an important pathogenic organism that causes intestinal and extraintestinal infections. Amongst the very common extraintestinal pathogenic E. coli are uropathogenic E. coli (UPEC). UPEC are usually classified in B2 phylogenetic group and are responsible for 80 % of urinary tract infections (UTI). Most of UPEC strains have gene for Usp — uropathogenic specific protein. Usp is nuclease pyocins and colicins homolog and has DNase activity in vitro. At first it was anticipated that Usp is a bactericide, but later it was shown that it does not have bactericidal effect on bacteria, but a genotoxic effect on eukaryotic cells — therefor Usp is a genotoxin. Downstream of usp gene there are three genes for Imu proteins, which protect producing cells from Usp nuclease activity. Imu3 reversibly binds DNA and promotes genotoxic effect of Usp protein. The aim of this master’s thesis was to prepare Usp-GFP fusion recombinant protein with activity similar to the native Usp protein activity. Usp-GFP production was optimized. We grew E. coli strain BL21 (DE3) pLyseE transformed with pTHCG12U on 30 °C and induced Usp-GFP protein overexpression with 0.1 mM IPTG. Protein was then isolated with Ni-NTA affinity chromatography and additionally purified with FPLC. Activity was tested afterwards. Imu3 and Usp isolates were also prepared. We showed that Usp-GFP has DNase activity and effect on HUVEC cells biomass amount. Usp-GFP in vitro and in vivo activity is comparable to native Usp activity. Usp-GFP protein is a useful tool for further research of interaction between Usp and eukaryotic cells.

• Keywords: Escherichia coli, uropathogenic specific protein, Usp, genotoxin, protein isolation, Imu3 protein, Usp-GFP